BIOAVAILABILITY OF REVERSIBLY SORBED ANDDESORPTION-RESISTANT 1,3-DICHLOROBENZENE

FROM A LOUISIANA SUPERFUND SITE SOIL

SANGJIN LEE1, R. R. KOMMALAPATI2,∗, K. T. VALSARAJ4, J. H. PARDUE1,3

and W. D. CONSTANT3

1Louisiana Water Resources Research Institute, Lousiana State University, Baton Rouge,LA 70803, U.S.A.; 2Department of Civil Engineering, PO Box 4249, Prairie View A&M University,

Prairie View, TX, U.S.A.; 3Department of Civil and Environmental Engineering Louisiana StateUniversity, Baton Rouge, LA 70803, U.S.A.; 4Gordon A. and Mary Cain Department of Chemical

Engineering, Louisiana State University, Baton Rouge, LA 70803, U.S.A.(∗author for correspondence, e-mail: r kommalapati@pvamu.edu, Fax: 9368572418,

Tel: 9368574125)

(Received 7 January 2003; accepted 24 March 2004)

Abstract. Microcosm batch studies were conducted to study the biodegradation of 1,3-dichlorobenzene (1,3-DCB) from the aqueous (soil free) and soil phases. For soil phase experiments,a freshly contaminated soil and a soil containing only the desorption-resistant or irreversibly boundor non-labile fraction of the contaminant were used. These experiments were designed to simulatebiodegradation at Superfund site assuming sorption/desorption equilibrium was reached. The pres-ence of the soil reduced the rates of biodegradation significantly. Nearly 100% of the total 1,3-DCBin the aqueous phase was biodegraded by enriched bacterial cultures within 7 days compared toabout 55% over a 6-week incubation period from the freshly contaminated soil. The biodegradationin the soils containing only the desorption-resistant fraction of the contaminant was considerablylower (about 30%). It is believed that for freshly contaminated soil, 1,3-DCB readily desorbed intothe aqueous phase and was available for microbial consumption whereas for soils containing mostlythe desorption-resistant fraction of 1,3-DCB, the contaminant availability was limited by the masstransfer into the aqueous phase. Our earlier studies concluded that about 20–30% of the sorbed con-taminant is tightly bound (even larger for weathered or aged soils) and is not easily extractable.This fraction is typically present in micropores or chemically bound to soil humic matter and thusis not readily accessible for microbial utilization. The findings presented here for 1,3-DCB are inagreement with those reported for other chemicals in the literature and could have implications tothe current remedy, the monitored natural attenuation at the Petro Processors Inc. Superfund site inLouisiana.

Keywords: dichlorobenzene, desorption, biodegradation, labile, non-labile, bioavailability

1. Introduction

Hydrophobic organic contaminants (HOCs) usually exhibit slow release rates fromsoil and sediment (Steinberg et al., 1987; Connaughton et al., 1993; Valsaraj et al.,1999; Lee et al., 2002). Recent research shows that HOCs, particularly aromaticcompounds, may be biodegraded by microorganisms to a residual concentrationthat no longer decreases or which decreases slowly over years with continued

Water, Air, and Soil Pollution 158: 207–221, 2004.C© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

208 S. LEE ET AL.

treatment (Linz and Nakles, 1997). It is widely believed that further reductionsare limited by the availability of hydrocarbons to microorganisms and all the moreso for aged contaminants as compared to freshly added material (Bosma et al.,1997). Zhang and Bouwer (1997) studied the bioavailability of benzene, tolueneand naphthalene in soil-water slurry, and noted that the rate of biodegradationdecreased with increasing organic compound hydrophobicity, soil/water ratio, soilparticle size, and soil organic carbon content suggesting that the bioavailability ofcontaminant was affected by soil characteristics and rate of sorption.

The bioavailability of a chemical is controlled by a number of physicochemicalprocesses such as sorption and desorption, diffusion, and dissolution (Ogram et al.,1985; Chung et al., 1993; Luthy et al., 1994; Zhang et al., 1998). In particular, foraged contaminated soils a fraction of the contaminant appears to be inaccessible forbiodegradation. All evidence seems to indicate a reduced availability of contaminantin soils and sediments contaminated for a prolonged period of time, and pollutant,not nutrient availability being the cause. This aging or weathering as it is usually re-ferred to, may result from (i) chemical oxidation reactions incorporating them intosoil organic matter (ii) slow diffusion into very small pores and adsorption into or-ganic matter or (iii) the formation of semi-rigid films around non-aqueous phase liq-uids with a high resistance toward organic-water mass transfer (Bosma et al., 1997).

Several researchers have confirmed that biodegradation can be limited by theslow desorption of organic compounds (Pignatello, 1989; Steinberg et al., 1987;Robinson et al., 1990; Al-Bashir et al., 1994; Lee et al., 2003). Robinson et al.(1990) observed that although most of the toluene in soil–water slurries was biode-graded rapidly, a small fraction was biodegraded much more slowly and at a ratelimited by desorption. In addition, the influence of sorption on biodegradation wasquantified by defining a bioavailability factor, B f (Zhang et al., 1998). However,whether this predictive modeling can explain the processes in complicated hetero-geneous natural systems remains to be verified. Steinberg et al. (1987) reported thatethylene dibromide (1,2-dibromoethane), a soil fumigant with relatively high wa-ter solubility, volatility and biodegradability persisted in top soils for as long as 19years after its last application. However, laboratory experiments with freshly addedcontaminant showed rapid biodegradation (Scribner et al., 1992). It is reportedthat residues of ethylene dibromide in the top few centimeters were unavailable formicrobes because it was sorbed into soil micropores and unavailable for biodegrada-tion. Salkinoja-Salonen et al. (1989) reported that no degradation of chlorophenolswas observed in soil that was polluted for over 40 years while chlorophenol degrada-tion proceeded rapidly in freshly polluted soil. A freshly added pentachlorophenol inthe polluted soil, however, was mineralized instantaneously suggesting that enoughchlorophenol-degrading bacteria and nutrients are available for the metabolism. Re-cently, some studies have reported degradation of desorption-resistant HOCs, suchas naphthalene and dichlorobenzene in soils (Park et al., 2001; Lee et al., 2003).

Though significant research has been conducted to study the sorption anddesorption kinetics of organic compounds and its bioavailability, few studies

BIOAVAILABILITY OF 1,3-DICHLOROBENZENE 209

have focused on the bioavailability of contaminants in soils containing only thedesorption-resistant fraction and how the degradation rates will compare to thosefor freshly contaminated soils. The work presented herein is part of an ongoing re-search supporting the remediation activities at a Louisiana Superfund site known asPetro Processors Inc. (PPI) located north of Baton Rouge, LA. In our earlier papers,we reported the adsorption/desorption hysteresis, and desorption kinetics for someof the prevalent contaminants at the site (Valsaraj et al., 1999; Kommalapati et al.,2002; Lee et al., 2002). Trichloroethylene and 1,3-dichlorobenzene desorption ex-hibited a biphasic desorption pattern with one fraction that is readily desorbed anda second fraction that was resistant to desorption. Also, the age of contaminationhas a significant effect on the fraction that is readily desorbed (Lee et al., 2002).

Several studies have reported on the degradation of DCBs in the aqueous phaseusing mixed and pure cultures (Reineke and Knackmuss, 1984; deBont et al., 1986;Jackson and Pardue, 1999). In this study, we conducted aerobic microcosm batchexperiments with aqueous phase (soil free) and soil–water slurries to determine theextent of 1,3-DCB biodegradation over a 6-week period. Another soil that containedonly the desorption resistant fraction (i.e., soil for which the readily desorbedfraction is removed in sequential desorption steps) was also used to study thebioavailability of the contaminant. The results of the study may have implicationsas evidence is being gathered to address the issues such as “how clean is clean?”and “what are the acceptable end points?”

2. Materials and Methods

2.1. SOIL

An uncontaminated soil from the Brooklawn site (one of the two sites known asPPI sites), located north of Baton Rouge, LA was used. Large lumps of soil werebroken and oven dried at 55 ◦C for 24 h. The soil was then pulverized before sievingthrough a 150 µm sieve (U.S. standard sieve no. 100). Samples from this soil weremethanol-extracted and analyzed and found to be free of the test contaminant, 1,3-dichlorobenzene, (1,3-DCB). The LSU Soil Science Laboratory performed soilanalysis as per the standard methods and the soil characteristics are presented inTable 1. This soil is classified as silty soil. All the soil samples were autoclavedbefore using them in the biodegradation experiments.

2.2. CHEMICALS

The test contaminant selected for this study was 1,3-dichlorobenzene (1,3-DCB)for which we also studied the adsorption/desorption kinetics with the same soil(Lee et al., 2002). 1,3-DCB with 98% purity was purchased from Aldrich Chemi-cal Co. (Milwaukee, WI) and was used as supplied. Aqueous solubility of 1,3-DCBis 123 mg L−1 at 25 ◦C, and its octanol–water partition coefficient (log Kow) is 3.60

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TABLE I

Physicochemical characteristics of thesoil used in the study

Characterization of soil Silty soil

Texture analysisClay (%) 10Silt (%) 82.2Sand (%) 7.9Organic Carbon 1.35Hydrogen (%) 0.33Oxygen (%) 2.01

Elemental analysisNitrogen (%) 0.06Sulfur (%) 0.04Ash (%) 96.38

(Montgomery, 1997). Sodium azide, NaN3 (Sigma Chemical Co., St. Louis, MO)was used to serve as a bacteriological inhibitor or biocide. The mineral salts mediumwas used for all the biodegradation experiments and it contained the following in-gredients (in g L−1): NH4Cl (0.1); MgSO4·7H2O (0.1); K2HPO4 (1.55), NaH2PO4–2H2O (0.85) and 2 ml of trace mineral solution (Kim, 1998).

2.3. GLASSWARE

Trace Clean R© bottles of 125 ml capacity (VWR Scientific, Sugar Land, TX) whichwere certified to be free of trace organics were used for this study. The caps wereblue, polypropylene open-top caps with bonded Teflon fluorocarbon resin/siliconsepta. More importantly these bottles can be used directly with the centrifuge atlow speeds and thus eliminate the need for transfer of sample and any volatilizationlosses associated.

2.4. BIOAVAILABILITY STUDIES

Biodegradation experiments were conducted in three different sets using (i) aqueousphase that is representative of soil porewater, (ii) freshly contaminated soil and (iii)soil containing only the desorption-resistant fraction of the contaminant obtainedby sequential desorption of a freshly contaminated soil.

2.4.1. Enrichment of 1,3-DCB-Degrading Bacterial CultureAerobic enrichment cultures were prepared with mixed cultures obtained fromsludge samples provided by a wastewater treatment plant (Baton Rouge, LA). About100 ml of mineral salts media and predetermined amount of 1,3-DCB stock solu-tion to give a final aqueous concentration of 200 µg L−1 were added to 250 ml

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Erlenmeyer flask and equilibrated under aerobic conditions at room temperature(22 ∼ 24 ◦C). Potassium phosphate buffer solution was added to maintain the pH ataround 7.0. Biological growth was monitored by measuring the volatile suspendedsolids concentration and turbidity with a UV–VIS Spectrophotometer (Shimadzu,Model UV-1201) as recommended by the Standard Methods (Eaton et al., 1995).Aqueous 1,3-DCB concentrations were measured using GC–MS to monitor thedegradation. The culture thus developed was used during its growth phase as theseed inoculum for all the biodegradation experiments.

2.4.2. Preparation of Contaminated Soil and Soil Wash Waterfor Biodegradation of 1,3-DCB

A measured amount (250 g) of dried uncontaminated soil was added to a 1 Lglass jar with Teflon R© lined cap. Aqueous solution spiked with 1,3-DCB (2 mgL−1) was added to these jars and mixed vigorously to wet the entire soil andthen filled to capacity so as to have minimum headspace. The jars were equili-brated on a tumbler for 72 h at 80 rpm. After the equilibration period, the jarswere left standing for about 1 day to obtain a clear soil–water interface, then theaqueous phase and the contaminated soil were separated and saved for furtheruse.

For the experiments using freshly contaminated soil, the soil obtained in theabove step was used directly after determining the soil concentration of 1,3-DCB.However, for experiments with soil containing only the desorption-resistant fractionof 1,3-DCB, the above soil was subjected to sequential desorption as described inour earlier papers (Lee et al., 2002; Valsaraj et al., 1999; Kommalapati et al.,2002). After separating the aqueous phase from the soil as mentioned in the earliersection, the jar containing the contaminated soil was filled with distilled water andequilibrated for 24 h. The aqueous phase was replaced again with distilled waterand the process was repeated four more times (a total of five desorption steps).From our earlier experiments, we have found that the contaminant in this caseremaining on the soil after five desorption steps can be assumed to be desorption-resistant. The soil concentration of 1,3-DCB was measured and the soil was usedfor the biodegradation experiments. The aqueous phase or the pore water collectedfrom the initial contamination step and the subsequent desorption steps was stored,analyzed for 1,3-DCB and used for the aqueous phase biodegradation studies afteradding appropriate amendments.

2.4.3. Biodegradation of 1,3-DCB in Aqueous Phase (Soil Porewater)The soil wash water obtained as above was analyzed for 1,3-DCB. About 10 mlof mineral salts medium prepared as mentioned earlier was added with 40 mlof soil porewater to the 125 ml Trace Clean bottles. A predetermined amount of1,3-DCB methanol stock solution (1000 mg L−1) was added to obtain an initial1,3-DCB aqueous concentration of about 5 mg L−1. About 5 ml of DCB-degradingbacterial culture was added to each bottle. However, for control experiments, 15 ml

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of sodium azide solution (200 mg L−1) was added instead of the nutrient solutionand seed inoculum. The total volume of the aqueous phase was about 55 ml forthe aqueous phase biodegradation studies. A total of 42 bottles (21 controls and21 with bacterial cultures) were equilibrated on a tumbler at 80 rpm and at roomtemperature. At predetermined time intervals three bottles containing the bacterialcultures and three control bottles were sacrificed and analyzed for 1,3-DCB. Theexperiment was continued for about 1 week.

2.4.4. Biodegradation of 1,3-DCB in Freshly Contaminated SoilThe contaminated soil obtained after equilibration of uncontaminated soil with 2mg L−1 of aqueous 1,3-DCB solution as mentioned above was used for this study.At the end of the equilibration period, contaminated soil was separated and analyzedfor 1,3-DCB. Approximately 7 g of this wet-contaminated soil was added into each125 ml Trace Clean bottle. Extreme care was taken during the soil transfer to preventpossible volatilization losses. About 25 ml of nutrient solution prepared as aboveand 26 ml of DCB-degrading bacterial culture was added to each bottle. For controlbottles, however, 26 ml of biocide, sodium azide (NaN3) solution (200 mg L−1)was added instead of bacterial culture. A total of 28 bottles (7 controls and 21 withbacterial cultures) were equilibrated on a tumbler at 80 rpm and at room temperature.At predetermined time intervals, three bottles containing the bacterial cultures andone control were sacrificed and analyzed for total 1,3-DCB after sonication removalwith 10 ml methanol. The experiment was continued for about 4 weeks.

2.4.5. Biodegradation of 1,3-DCB in Soil Containing only Desorption-ResistantFraction

The contaminated soil subjected to five sequential desorption steps was used for thisstudy. The procedure followed is exactly the same as that for freshly contaminatedsoil described above.

2.5. EXTRACTION AND ANALYSIS OF 1,3-DCB

Extraction of 1,3-DCB from the test bottles containing soil and the aqueous solu-tions was done using a sonication technique. Appropriate amounts of HPLC grademethanol (10 ml) were added to the bottles and sonicated for 30 min. The aqueoussolution was separated after centrifugation and the supernatant was analyzed forDCB.

Aqueous solutions and the soil extracts which contained methanol and waterwere analyzed for 1,3-DCB using a purge-and-trap liquid sample concentrator(Tekmar, Model LSC-2) attached to a HP 5890A gas chromatograph equipped witha HP5971 mass selective detector (MS) and an autosampler. The samples werepurged with helium gas. The total run time for the method was 20 min and themethod detection limit (MDL) was 1µg L−1.

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3. Results and Discussion

3.1. DESORPTION OF 1,3-DCB

We reported that the desorption of hydrophobic organic compounds from soil ex-hibits biphasic behavior namely a readily sorbing or labile phase also known asa reversible fraction and another phase containing desorption-resistant or tightlybound or non-labile fraction (Valsaraj et al., 1999; Kommalapati et al., 2002; Leeet al., 2002). We used an empirical model developed by Opdyke and Loehr (1999)to describe our experimental observations.

St

So= 1 − Fe−k1t − (1 − F)e−k2t , (1)

where t is time (h), St/S0 is the fraction of chemical released after time t , F isthe fraction of chemical released quickly (reversibly bound/labile fraction) and(1 − F) is the fraction of chemical released slowly (irreversibly bound/non-labile/desorption-resistant fraction). k1 and k2 are the first-order rate constantsdescribing the desorption of the labile fraction and slowly released (non-labile)fraction (h−1). The experimental data obtained for 1,3-DCB with silty soilfrom PPI site was fitted to the above equation to determine the three unknownmodel parameters by using Curve Expert (Version 1.34, a free download fromhttp://www.ebicom.net/∼dhyams/cvxpt.htm). The labile fraction of the contam-inant (F) is determined to be 0.6 with rate constants k1 and k2 to be 0.28and 6 × 10−4 h−1, respectively (Lee et al., 2002). Figure 1 shows the desorp-tion kinetics as a function of time for 1,3-DCB. The line is obtained using themodel parameters determined from the curve fit. It was clear from the figurethat 70% of the sorbed 1,3-DCB was desorbed in about 5 days and the remain-der (30%) was still sorbed to soil. This fraction is usually referred in the litera-ture as desorption-resistant or non-labile or irreversibly bound or tightly boundfraction.

Soil containing only the desorption-resistant fraction was obtained for this studyby following the protocol developed in our earlier study (Lee et al., 2002). Thefreshly contaminated soil was subjected to sequential desorption. The contaminantremaining after five desorption steps was considered desorption-resistant and usedfor the experiments. Figure 2 shows a plot of the soil phase concentration and thecorresponding aqueous concentration during the sequential desorption. The firstpoint on the plot corresponds to the initial equilibrium sorption (soil contamination)step followed by the five points that correspond to the five successive desorptionsteps. As seen from the figure, after five desorption steps, most of the readilydesorbed fraction of 1,3-DCB was removed from soil. This is in agreement withearlier reports (Di Toro and Horzempa, 1982; Valsaraj et al., 1999; Lee et al.,2002). The initial 1,3-DCB concentration was about 14,400 µg kg−1 soil for freshly

214 S. LEE ET AL.

Figure 1. Desorption kinetic data showing fraction removal as a function of time for 1,3-DCB ona PPI site soil silty soil. The lines were obtained using the model parameters determined using thecurve fit.

Figure 2. Sequential desorption of 1,3-DCB from soil. The arrows point to the soils that were usedfor the biodegradation experiments.

BIOAVAILABILITY OF 1,3-DICHLOROBENZENE 215

contaminated soil compared to about 1650 µg kg−1 soil for the experiments usingsoil containing the desorption-resistant fraction of the contaminant.

3.2. BIODEGRADATION OF 1,3-DCB IN THE AQUEOUS PHASE

Aqueous phase biodegradation of 1,3-DCB was studied using soil pore water ob-tained from the desorption of DCB from freshly contaminated soil. To maintain suf-ficient oxygen levels, 0.05 ml of hydrogen peroxide, which is equivalent to 9.63 mgL−1 of oxygen was added into each vial. Mixed cultures acclimatized with 1,3-DCBwere used as seed inoculua for the microcosm studies. Extreme care was taken tominimize losses due to volatilization and other abiotic processes. Our experimentaldata from the control samples indicated negligible losses as seen from little variationin the initial aqueous phase concentration (about 5 mg L−1). Biodegradation of 1,3-DCB was monitored by sacrificing triplicate samples at predetermined intervals.

Aqueous phase concentrations of 1,3-DCB in both the control samples and thetreatment samples are shown in Figure 3. The averages from the triplicate samplesare used for the plots. The biodegradation kinetic data for 1,3-DCB was fitted to afirst-order decay model. The aqueous phase biodegradation as expected provided abetter fit to the decay model compared that for the soil phases (Table II). The k valuesestimated from the regression were compared at 95% confidence interval. The k,decay constant, was estimated to be 0.0212 h−1 along with about 32 h of a half-life

Figure 3. Biodegradation of 1,3-DCB in aqueous phase. Percent degradation was calculated afteraccounting for the losses in the controls.

216 S. LEE ET AL.

TABLE II

Kinetic parameters of first-order kinetics in 1,3-dichlorobenzene (1,3-DCB)

Treatments K (h−1)a T1/2 (h) R2

Aqueous phase 0.0212 (0.0014) 32.69 0.99

Freshly contaminated fractionin soil phase

0.0009 (0.0003) 770 0.82

Desorption resistant fractionin soil phase

0.0002 (0.0001) 3465 0.65

aValues in parentheses are standard errors of parameter estimates.

in inoculated solution. Disappearance of 1,3-DCB in solution was significantlyenhanced after inoculation of 1,3-DCB-degrading bacteria when compared to theuninoculated control (Figure 3). As can be seen from the figure, the 1,3-DCBconcentrations decreased to about 1 mg L−1 (about 80% degradation) in 3 daysand nearly complete biodegradation was accomplished within 7 days. It shouldbe noted that our preliminary studies showed only 80% degradation and didn’tchange appreciably even after extended periods of incubation. The oxygen depletionseemed to be responsible and the addition of hydrogen peroxide to the sample vialsprovided enough oxygen thus making the microbial growth substrate limited. Thegrowth and metabolism of microorganisms is sustained for the duration of theexperiment as indicated by the protein concentration (second y-axis on the plot).The protein concentration, which represents the growth of the cultures, increasedsteadily and reached a maximum around 120 h and remained constant at about18 mg L−1 for the remainder of the experiments or in other words, the cultures arein the stationary phase. These observations are in agreement with those reportedin the literature for DCB and other similar compounds (Reineke and Knackmuss,1984; deBont et al., 1986; Jackson and Pardue, 1999).

Several researchers have noted that the rate of biodegradation in the aqueousphase (or soil free) was faster than that in the presence of soil (Robinson et al., 1990;Zhang and Bouwer, 1997). Our results of the soil phase experiments discussed inthe following sections corroborate these observations. The initial concentration of1,3-DCB is lower in our studies compared to those reported in the literature.

3.3. BIODEGRADATION OF 1,3-DCB IN FRESHLY CONTAMINATED SOIL

Soil for these experiments was obtained by equilibrating an uncontaminated soilwith aqueous solution spiked with 1,3-DCB followed by separation of the aque-ous phase. The soil thus obtained was analyzed for DCB concentration and usedin the microcosms directly. The soil was amended with nutrients and inoculatedwith the seed cultures. Figure 4 shows the percent 1,3-DCB degraded as a func-tion of incubation time for freshly contaminated soil and soil containing only the

BIOAVAILABILITY OF 1,3-DICHLOROBENZENE 217

Figure 4. Biodegradation of 1,3-DCB from freshly contaminated soil and soil containing onlydesorption-resistant fraction of 1,3-DCB. Percent degradation was calculated after accounting forthe losses in the controls.

desorption-resistant fraction. Again the data points are obtained by averaging trip-licate samples. The percent degradation presented in the plot accounts for all thelosses reported in the control samples. The losses in the controls were less than10%, which is very reasonable for complex systems such as this one. As can beseen from the figure, the percent biodegradation was 23% after the first day ofincubation in the microcosm. Thereafter, the biodegradation increased slowly butsteadily as indicated by the positive slope in the plot to about 55% over the 6-weekincubation period. The positive slope of the biodegradation curve is also an indica-tion that neither oxygen nor nutrients were limiting the growth. It is possible that thedegradation of 1,3-DCB will proceed, albeit at a very slow rate, if the experimentswere continued beyond the 6-week period. It was noted in our earlier studies thatabout 60% (F = 0.6 from Equation (1)) of the sorbed contaminant, 1,3-DCB isreversibly bound and the rest being irreversibly bound to soil or in other words inthe desorption-resistant fraction (Lee et al., 2002; Valsaraj et al., 1999). It appearsfrom the figure, that bacteria were able to degrade a significant portion of the readilyavailable fraction of 1,3-DCB.

As would be expected, the rate of biodegradation of DCB was much slower inthe soil phase than that in the aqueous phase. For example, nearly 80% of 1,3-DCBwas biodegraded within 3 days of incubation and nearly complete degradation in7 days for the aqueous phase (soil free) compared to about 55% degradation from

218 S. LEE ET AL.

freshly contaminated soil. This is in agreement with the findings from the literature(Zhang and Bouwer, 1997; Robinson et al., 1990). The decay of 1,3-DCB overtime was fitted to a first-order decay model as well, but the model fit for the soilphase did not provide a very high correlation coefficient compared to that for theaqueous phase (Table II). The k, decay constant, was estimated to be 0.0009 h−1

which was less than 1/20th of that for aqueous phase. The half-life was greater than770 h in inoculated solution. Disappearance of 1,3-DCB in the presence of soil wassignificantly inhibited compared to aqueous phase (Figure 4). It is also reportedthat microorganisms utilize the substrate (contaminant) from the aqueous phaserather than metabolize the contaminant directly sorbed to the soil. Hence masstransfer of DCB from soil surface to the aqueous phase could play a significantrole and potentially limit biodegradation (Robinson et al., 1990; Chung et al.,1993; Zhang et al., 1998; Lee et al., 2003). However, for the readily desorbingfraction the mass transfer (desorption) does not seem to limit the biodegradationthough it may control the rate. However, this will not be the case for experimentswith the soil containing desorption-resistant fraction as discussed in the followingsection.

3.4. BIODEGRADATION OF 1,3-DCB FROM SOIL CONTAINING

THE DESORPTION-RESISTANT FRACTION

A known amount of the soil that was contaminated as above and subjected to sequen-tial desorption and containing only the desorption-resistant fraction of DCB wasadded to microcosms, which were prepared as above. The biodegradation of 1,3-DCB was monitored with time over a 6-week incubation period. As noted earlier, theinitial soil concentration was about 1650 µg kg−1 soil compared to 14,400 µg kg−1

soil for freshly contaminated soil experiments. The percent biodegradation wascalculated as above after accounting for the losses from the control samples.Figure 4 also shows the percent biodegradation as a function of incubation time forthe soil containing the desorption resistant fraction of 1,3-DCB. As can be notedfrom the figure, for the soil containing the desorption-resistant fraction DCB thedegradation was significantly lower. For example, 1,3-DCB degradation was about22% during the first week with only a total of 33% during the total 6-week incu-bation period. The estimated k, decay constant, is 0.0002 h and a half-life of 3465h, which is more than four times longer than that for freshly contaminated soil.It is apparent that mass transfer from the soil to the aqueous phase is limiting thebiodegradation of DCB in soils. It should be noted that triplicate samples were sac-rificed at each sampling period and there are slight variations among the triplicates.The soil used in the experiments was subjected to five sequential desorption stepsto remove the readily desorbing fraction before being used for the biodegradationstudies. It should however be kept in mind that five desorption steps may not re-move all of the readily desorbing fraction from soil and thus it is possible a smallfraction may still be readily available for the microbes during the initial incubation

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period as indicated by the initial high rate of biodegradation. As expected, 1,3-DCBbiodegradation rates were reduced significantly after the first week. The desorptionfrom the desorption-resistant fraction is limiting the availability of the contaminantfor biodegradation. The microorganisms metabolize the substrate present in theaqueous phase rather than direct metabolism from the soil phase. Though there hasbeen some literature to suggest that sorbed substrate may be directly available fordegradation by attached cells either by direct partitioning to the cell membrane orvia degradation by extracellular enzymes (Park et al., 2001), there is still no clearagreement on the overall degradation mechanism.

Figure 4 also provides a comparison between freshly contaminated soil and soilwith only a desorption-resistant fraction. As evident from the figure, the degra-dation in soil containing desorption-resistant contaminant was 30% compared toabout 55% for freshly contaminated soil. A number of investigators have shown thatthe desorption-resistant fraction in the soil is unavailable for microorganisms evenunder optimal conditions for biodegradation (Zhang and Bouwer, 1997; Robinsonet al., 1990; Al-Bashir et al., 1994; Pignatello, 1989). The irreversibly bound ordesorption-resistant fraction of the contaminant could be present in the soil microp-ores or chemically bound to soil humic matter and thus microorganisms may not beable to access this fraction. This is supported by data from Robinson et al. (1990)who observed that acclimated bacteria readily degraded the extractable fractionof sorbed toluene, which accounted for about 90% of the total toluene. However,a small unextractable portion biodegraded at a rate limited by desorption. Ogramet al. (1985) reported that acclimated bacteria could biodegrade only the aque-ous phase contaminant and could not utilize the sorbed phase contaminant. Ourmicrocosm batch studies showed that microorganisms readily degraded the easilyextractable or readily desorbing fraction of the sorbed DCB and that desorptionmass transfer rates are limiting the growth of the microorganisms and thus the rateof biodegradation.

4. Conclusions

The bioavailability of 1,3-DCB from freshly contaminated soils and soils containingonly a desorption-resistant fraction was studied along with that in the aqueousphase. In the aqueous phase, 1,3-DCB was rapidly degraded and degradation wassignificantly inhibited in the presence of soil, and the effect was likely caused bysorption to the solid phase. About 70% of the total 1,3-DCB was degraded withinthe 4-week incubation period for freshly contaminated soils compared to about20% for soils containing desorption-resistant 1,3-DCB. Biodegradation rates inthe soil phase were significantly slower in the desorption-resistant fraction thanrates in freshly contaminated fraction. 1,3-DCB half-life in the presence of soilwas dramatically increased to 770 and 3465 h for freshly contaminated soil andsoil containing desorption-resistant fraction compared to about 32 h for aqueousphase. The decay constants were 0.0212 h−1 for aqueous phase biodegradation

220 S. LEE ET AL.

compared to 0.0009 for freshly contaminated soil and 0.0002 for soil containingdesorption-resistant fraction. From our earlier desorption studies, we reported thatabout 20–30% of the sorbed contaminant is in the desorption-resistant compartmentand is not easily extractable. This available fraction could be even smaller for aged orweathered soils. It appears from our microcosm batch studies that microorganismscan readily degrade the easily extracted fraction of the sorbed DCB and desorptionmass transfer rates are limiting the growth of the microorganisms and thus theoverall biodegradation. Thus, if the desorption-resistant fraction is not available forbiodegradation and is not extracted with water as found in a pump and treat (P&T)technology, it brings up an issue whether it is still harmful to human health and theenvironment. Further, cleanup goals and hence endpoints need to be assessed on acase-by-case basis depending on the mobility and bioavailability of chemicals ofconcern at a specific site.

Acknowledgement

This work was supported by the LSU Hazardous Substance Research Center/South& Southwest with sponsorship of the U.S. District Court, Middle District ofLouisiana and support from NPC Services, Inc.

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